1 Taxon sampling and inferences about diatom phylogeny

Proper taxon sampling is one of the greatest challenges to understanding phylogenetic relationships, perhaps as important as choice of optimality criterion or data type. This has been demonstrated in diatoms where centric diatoms may either be strongly supported as monophyletic or paraphyletic when analyzing SSU rDNA sequences using the same optimality criterion. The effect of ingroup and outgroup taxon sampling on relationships of diatoms is explored for diatoms as a whole and for the order Thalassiosirales. In the latter case, SSU rDNA and rbcL sequence data result in phylogenetic relationships that appear to be strongly incongruent with morphology and broadly incongruent with the fossil record. For example, Cyclotella stelligera Cleve & Grunow behaves like a rogue taxon, jumping from place to place throughout the tree. Morphological data place C. stelligera near the base of the freshwater group as sister to the extinct genus Mesodictyon Theriot and Bradbury, suggesting that it is an old, long branch that might be expected to “misbehave” in poorly sampled trees. Cyclotella stelligera and C. bodanica Grunow delimit the diameter of morphological diversity in Cyclotella, so increased sampling of intermediate taxa will be critical to resolving this part of the tree. Morphology is sampled for a much greater number of taxa and many transitional states of putative synapomorphies seem to suggest a robust morphological hypothesis. The Thalassiosirales are unstable with regards to taxon sampling in the genetic data, suggesting that perhaps the morphological hypothesis is (for now) preferable.     

Effects of intermediate filaments on actin-based motility of Listeria monocytogenes.

How does subcellular architecture influence the intracellular movements of large organelles and macromolecular assemblies? To investigate the effects of mechanical changes in cytoplasmic structure on intracellular motility, we have characterized the actin-based motility of the intracellular bacterial pathogen Listeria monocytogenes in normal mouse fibroblasts and in fibroblasts lacking intermediate filaments. The apparent diffusion coefficient of L. monocytogenes was two-fold greater in vimentin-null fibroblasts than in wild-type fibroblasts, indicating that intermediate filaments significantly restrict the Brownian motion of bacteria. However, the mean speed of L. monocytogenes actin-based motility was statistically identical in vimentin-null and wild-type cells. Thus, environmental drag is not rate limiting for bacterial motility. Analysis of the temporal variations in speed measurements indicated that bacteria in vimentin-null cells displayed larger fluctuations in speed than did trajectories in wild-type cells. Similarly, the presence of the vimentin meshwork influenced the turning behavior of the bacteria; in the vimentin-null cells, bacteria made sharper turns than they did in wild-type cells. Taken together, these results suggest that a network of intermediate filaments constrains bacterial movement and operates over distances of several microns to reduce fluctuations in motile behavior.     

Mechanisms of MAdCAM-1 gene expression in human intestinal microvascular endothelial cells

Mucosal addressin cell adhesion molecule-1 (MAdCAM-1) is a homing receptor preferentially expressed on gut-associated endothelial cells that plays a central role in leukocyte traffic into the mucosal immune compartment. Although the molecular mechanisms underlying endothelial ICAM-1 or E-selectin expression have been intensively investigated, the mechanisms that regulate human MAdCAM-1 expression have not been defined. We report MAdCAM-1 gene and protein expression in primary cultures of human intestinal microvascular endothelial cells (HIMEC) that was not demonstrated in human umbilical vein endothelial cells. Similar to ICAM-1 and E-selectin expression, MAdCAM-1 gene expression in HIMEC was inducible with TNF-, IL-1, or LPS activation. However, in striking contrast to ICAM-1 and E-selectin expression, MAdCAM-1 mRNA and protein expression in HIMEC was heavily dependent on culture duration and/or cellular density, suggesting a prominent role for cell-cell interaction among these endothelial cells in the expression of the mucosal addressin. MAdCAM-1 expression was inhibited by both SN-50 (NF-B inhibitor) and LY-294002 [phosphatidylinositol 3-kinase (PI3-K) inhibitor], whereas ICAM-1 and E-selectin expression was inhibited by SN-50 but not by LY-294002. The Akt phosphorylation by TNF- or LPS was greater at higher cell density, demonstrating a pattern similar to that of MAdCAM-1 expression. NF-B activation was not affected by cellular density in HIMEC. MAdCAM-1 expression in human gut endothelial cells is regulated by distinct signaling mechanisms involving both NF-B and PI3-K/Akt. These data also suggest that PI3-K/Akt is involved in the gut-specific differentiation of HIMEC, which results in expression of the mucosal addressin MAdCAM-1. cell adhesion molecules; nuclear factor-B; phosphatidylinositol 3-kinase     

Ena/VASP proteins contribute to Listeria monocytogenes pathogenesis by controlling temporal and spatial persistence of bacterial actin-based motility

The Listeria monocytogenes surface protein ActA mediates actin-based motility by interacting with a number of host cytoskeletal components, including Ena/VASP family proteins, which in turn interact with actin and the actin-binding protein profilin. We employed a bidirectional genetic approach to study Ena/VASP's contribution to L. monocytogenes movement and pathogenesis. We generated an ActA allelic series within the defined Ena/VASP-binding sites and introduced the resulting mutant L. monocytogenes into cell lines expressing different Ena/VASP derivatives. Our findings indicate that Ena/VASP proteins contribute to the persistence of both speed and directionality of L. monocytogenes movement. In the absence of the Ena/VASP proline-rich central domain, speed consistency decreased by sixfold. In addition, the Ena/VASP F-actin-binding region increased directionality of bacterial movement by fourfold. We further show that both regions of Ena/VASP enhanced L. monocytogenes cell-to-cell spread to a similar degree, although the Ena/VASP F-actin-binding region did so in an ActA-independent manner. Surprisingly, our ActA allelic series enabled us to uncouple L. monocytogenes speed from directionality although both were controlled by Ena/VASP proteins. Lastly, we showed the pathogenic relevance of these findings by the observation that L. monocytogenes lacking ActA Ena/VASP-binding sites were up to 400-fold less virulent during an adaptive immune response.     

The polymerization motor

Polymerization and depolymerization of actin filaments and microtubules are thought to generate force for movement in various kinds of cell motility, ranging from lamellipodial protrusion to chromosome segregation. This article reviews the thermodynamic and physical theories of how a nonequilibrium polymerization reaction can be used to transduce chemical energy into mechanical energy, and summarizes the evidence suggesting that actin polymerization produces motile force in several biological systems.     

Analysis of Surface Protein Expression Reveals the Growth Pattern of the Gram-Negative Outer Membrane

The outer membrane (OM) of Gram-negative bacteria is a complex bilayer composed of proteins, phospholipids, lipoproteins, and lipopolysaccharides. Despite recent advances revealing the molecular pathways underlying protein and lipopolysaccharide incorporation into the OM, the spatial distribution and dynamic regulation of these processes remain poorly understood. Here, we used sequence-specific fluorescent labeling to map the incorporation patterns of an OM-porin protein, LamB, by labeling proteins only after epitope exposure on the cell surface. Newly synthesized LamB appeared in discrete puncta, rather than evenly distributed over the cell surface. Further growth of bacteria after labeling resulted in divergence of labeled LamB puncta, consistent with a spatial pattern of OM growth in which new, unlabeled material was also inserted in patches. At the poles, puncta remained relatively stationary through several rounds of division, a salient characteristic of the OM protein population as a whole. We propose a biophysical model of growth in which patches of new OM material are added in discrete bursts that evolve in time according to Stokes flow and are randomly distributed over the cell surface. Simulations based on this model demonstrate that our experimental observations are consistent with a bursty insertion pattern without spatial bias across the cylindrical cell surface, with approximately one burst of ∼10⁻² µm² of OM material per two minutes per µm². Growth by insertion of discrete patches suggests that stochasticity plays a major role in patterning and material organization in the OM.     

Fine-scale time-lapse analysis of the biphasic, dynamic behaviour of the two Vibrio cholerae chromosomes

Using fluorescent repressor-operator systems in live cells, we investigated the dynamic behaviour of chromosomal origins in Vibrio cholerae, whose genome is divided between two chromosomes. We have developed a method of analysing fine-scale motion in the curved co-ordinate system of vibrioid bacteria. Using this method, we characterized two different modes of chromosome behaviour corresponding to periods between segregation events and periods of segregation. Between segregation events, the origin positions are not fixed but rather maintained within ellipsoidal caged domains, similar to eukaryotic interphase chromosome territories. These domains are approximately 0.4 microm wide and 0.6 microm long, reflecting greater restriction in the short axis of the cell. During segregation, movement is directionally biased, speed is comparable between origins, and cell growth can account for nearly 20% of the motion observed. Furthermore, the home domain of each origin is positioned by a different mechanism. Specifically, the oriC(I) domain is maintained at a constant actual distance from the pole regardless of cell length, while the oriC(II) domain is maintained at a constant relative position. Thus the actual position of oriC(II) varies with cell length. While the gross behaviours of the two origins are distinct, their fine-scale dynamics are remarkably similar, indicating that both experience similar microenvironments.